review article

Published online: 17 december 2009 | doi: 10.1038/nchem.473

Designing artificial enzymes by intuition

and computation
Vikas Nanda1* and Ronald L. Koder2

The rational design of artificial enzymes, either by applying physico–chemical intuition of protein structure and function or with
the aid of computational methods, is a promising area of research with the potential to tremendously impact medicine, indus‑
trial chemistry and energy production. Designed proteins also provide a powerful platform for dissecting enzyme mechanisms
of natural systems. Artificial enzymes have come a long way from simple α‑helical peptide catalysts to proteins that facilitate
multistep chemical reactions designed by state-of-the-art computational methods. Looking forward, we examine strategies
employed by natural enzymes that could be used to improve the speed and selectivity of artificial catalysts.

I
n the nineteen-fifties and sixties, the advent of the semiconductor
transistor and the integrated circuit transformed digital comput-
ers from powerful curiosities into pragmatic, cost-effective tools.
Along with advances in numerical methods, computers revolution-
ized the design and construction of aircraft, allowing engineers to
simulate complex, nonlinear systems that integrated aerodynamics,
propulsion, control and so on, thereby pushing aircraft technology
well beyond what was possible with previous analytical models.
Today, a Boeing 747 is an incredibly complex machine with over
6,000,000 parts. As such, computers have become indispensable in
the aerospace industry. Although much smaller in size, the mecha-
nistic complexity of enzymes and challenges associated with their
design (Box  1) suggest that they are as sophisticated as passenger
airliners, and it is expected that computational methods in chemistry
and biology will promote a similar revolution in the design of artifi-
cial catalysts.
The promise of constructing enzymes that are capable of effi-
ciently catalysing virtually any chemical reaction is a tremendous
motivator for researchers in the protein-design field. Enzymes cata-
lyse difficult chemical reactions in mild, aqueous environments,
often with a speed and specificity unrivalled by synthetic catalysts.
Designing an enzyme from scratch is also the most rigorous way of
testing our understanding of how natural enzymes function. Several
recent designs have been stripped-down or rebuilt versions of natu-
ral enzymes, providing powerful tools for dissecting molecular con-
tributions to enzyme structure and reactivity.
Enzyme design is inextricably linked with the design of protein
structure. Advances in protein design are often rapidly followed by
attempts to apply new technologies to artificial enzymes. Therefore,
this is as much a review of protein fold design as of catalyst design.
However, it should be noted that complex protein topologies are not
a prerequisite for catalysis. Proline alone can catalyse a remarkable
array of reactions, including aldolase-like formations of carbon–
carbon bonds through enamine intermediates with high yields and
substantial product enantiomeric excess. Other processes includ-
ing asymmetric epoxidations and acylations are achievable using
short peptides. The impressive catalytic properties of proline and
small peptides have been extensively reviewed previously 1,2 and are
not covered here. Few designed enzymes have achieved the cata-
lytic utility of such small peptides, and much remains to be done
before designer enzymes find practical applications. However, the
remarkable selectivity, rate-enhancements and product specificity
of natural enzymes under aqueous conditions warrants more work
in developing powerful molecular design technologies.
The complexities of enzyme design can be quite daunting.
Examining high-resolution structures of natural enzyme–substrate
complexes reveals that the conformations of active-site amino acids
are poised to facilitate catalysis. Second-shell interactions tune the
reactivity of the active site through networks of direct interactions with
primary ligands and long-range electrostatic forces. Designing such
molecules from scratch presents a host of computational challenges
(see Box 1). Accurate modelling of important forces in the active site
requires quantum mechanical (QM) calculations. Unfortunately, it is
not feasible to perform QM calculations on molecules the size of even
the smallest enzymes. Design also requires the rapid evaluation of a
large number of candidate structure/sequence combinations and QM
calculations are very demanding on computational resources. Effective
treatment of water molecules and their interactions with active-site
residues and reactants can also significantly increase the complexity
of calculations. Along with high-resolution design of the active-site
residues, candidate enzymes must maintain overall structural integ-
rity, and in some cases, incorporate large-scale protein motions that
may support catalysis. Integrating all of these factors into a design is a
formidable challenge.
It is reasonable to ask what one gains through such sophisticated
computation. After all, a number of novel folds and catalytically
active proteins have been built without such tools. In this review, we
survey several designed, artificial enzymes that have been developed
with varying degrees of computational involvement. These include
de novo enzymes, where both the protein topology and the active
site are built from scratch, and active-site design, where surfaces and
cavities on existing proteins are repurposed for catalysis. We build
on previous reviews of this field (for example, see ref. 3) by includ-
ing a deeper discussion of computational challenges associated with
enzyme design. Additionally, we look to the future of design, such as
introducing multiple substrates, protein motion, allostery into arti-
ficial enzymes, and expanding protein design principles to a broader
class of structured, catalytically active polymers.
The helix and the enzyme
A fundamental paradigm in biochemistry is the link between a
protein’s function and its three-dimensional fold, which in turn

1
Robert Wood Johnson Medical School - UMDNJ Biochemistry, Center for Advanced Biotechnology and Medicine, 679 Hoes Lane West, Piscataway, New
Jersey 08854, USA; 2City College of New York Physics, 160 Convent Avenue, New York, New York 10031, USA. *e-mail: nanda@cabm.rutgers.edu

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© 2010 Macmillan Publishers Limited. All rights reserved.
review article
Box 1 | Anatomy of an enzyme.
Hen egg white lysozyme (HEWL) was the first enzyme atomic
structure to be solved by X‑ray crystallography in 1965110. The
three-dimensional structure highlights many of the physical
characteristics of enzymes that make them unusually challeng-
ing proteins to design. HEWL functions in antibacterial defence
and cleaves glycosidic linkages found in bacterial cell walls. The
active site consists of two amino acids, a glutamic acid, which
functions as a general acid/base, and an aspartic acid nucle-
ophile. These are placed at the bottom of a deep substrate cleft,
which confers specificity and poises the substrate over the active
site. Many small-molecule catalysts function better in organic
solvents, where the low bulk dielectric enhances electrostatic
interactions. The cleft mimics this by isolating catalytic groups
from bulk water, strengthening local electrostatic interactions.
Accurate modelling of catalytic residue conformations and local
electrostatics are key in designing effective artificial enzymes.
Quantum mechanics methods have been useful in moving this
area of design forward.
The protein fold must be sufficiently stable to form this cleft
and preorganize active-site residues, which is one reason why
enzymes are much larger than synthetic catalysts. The computa-
tional design of proteins with partially buried polar active sites
is especially challenging. The protein fold must be able to absorb
the energetic cost of desolvating polar active-site groups and sta-
bilizing electrostatic interactions that favour catalysis.
Substrate cleft Water
Active site
Protein fold
depends on its amino acid sequence. Getting from sequence to
structure to function is the common goal unifying all work in the
field of protein design. Some of the earliest model proteins were
built with the α‑helix as the fundamental unit of structure. A seven-
residue repeating sequence, , where the first and fourth
amino acids are nonpolar ( ) and the rest polar ( ), will charac-
teristically form multimeric, left-handed coiled-coil assemblies of
amphipathic α‑helices; nonpolar side-chains associate between heli-
cal elements to form a hydrophobic core4–8. Specific homo and het-
erooligomers of α‑helices can be achieved by rational design of core
packing interactions and surface electrostatics9–11. The power of this
simple idea was demonstrated in a clever design where the super-
helical twist was inverted by changing the motif to an eleven-residue
repeat, , thus maintaining a continuous hydropho-
bic core in a right-handed coiled-coil12. This was an example of a
true de novo design with no known natural counterpart at the time
it was constructed.
The majority of early attempts to construct enzymes used
an amphipathic α‑helix as the principle structural component.
‘Helichrome’ was designed to function as a hydrolase, using four
α‑helices to form a hydrophobic substrate binding pocket over one
face of an iron porphoryn to which the helices were covalently teth-
ered13. Helichrome could convert aniline to p‑aminophenol in the
16 nature chemistry | VOL 2 | JANUARY 2010 | www.nature.com/naturechemistry
© 2010 Macmillan Publishers Limited. All rights reserved.
Nature chemistry doi: 10.1038/nchem.473
presence of NADPH with a kcat of 0.02 min–1 and Km of 5.0 mM. (For
most simple reactions, Km describes the affinity of the enzyme for
the substrate and kcat reflects the rate of product formation under
conditions where the enzyme is saturated by substrate.)
In work by Barbier and Brack, it was found that regular copoly-
mers of leucine and lysine could hydrolyse polyribonucleotides14,15.
Poly(Lys–Leu) and poly(Leu–Lys–Lys–Leu) had superior hydrolysis
rates to random copolymers or those incorporating both d,l-Leu
and d,l-Lys, demonstrating that either β‑sheet structure (formed by
a poly‑ pattern16) or α‑helical structure had a significant role in
improving activity. The regular structure provided a cationic surface
to support binding of negatively charged substrates such as nucleic
acids. Repulsive electrostatic interactions between adjacent lysine
side-chains served to depress the pKa of the amino group, making it
a better catalytic base.
These features were exploited in the design of ‘oxaldie’, a fifteen-
residue α‑helical peptide comprising mostly leucines and lysines
that catalysed decarboxylation of oxaloacetate to pyruvate through
an imine intermediate17 (Fig.  1). Possible amine donors in the
covalent intermediate were lysine side-chains and the backbone
amino terminus. The activity of the peptide was concentration
dependent, suggesting that a helical multimer was the active form.
Inserting a helix-breaking proline into the centre of the peptide
significantly reduced activity, affirming the relationship between
structure and activity. Rate enhancements of 103 to 104 over free
amines were observed.
Oxaldie was designed on the premise that the α‑helix would raise
the basicity of the active site, whether it was the amino terminus
through interactions with helix macrodipole, or the amino groups
of lysine through electrostatic interactions between side chains. This
made it possible to rationally improve activity by targeting struc-
ture. In one strategy, lysines were placed on the solvent-exposed
face of avian pancreatic peptide, a 36-amino-acid protein with a
helix packing against an extended polyproline chain. The resulting
oxaldie‑3 was monomeric, unlike its predecessor, eliminating the
need for high peptide concentrations to elicit catalysis18. Modest
improvements in kcat and Km were also achieved. In oxaldie‑4, a
bovine pancreatic peptide scaffold with intrinsic disulfides further
stabilized the fold, resulting in even better kinetic parameters and
activity at higher temperatures19. The same scaffold was adapted to
make a miniature esterase20.
The self-replicating peptides are an interesting case of α‑helical
enzymes where the catalyst does not form covalent intermediates
with reactants21,22. The enzyme is a 32-residue amphipathic helix
A B
a b c d e f g
Hy
ionic face
dro
phobic fa
g
C at
c
ce
d
f Oxaldie-1
a
b
e
Figure 1 | From sequence to structure to function. A, A repeating seven-
residue pattern of nonpolar (filled circles) and polar (open circles) residues
will create a hydrophobic surface on one face of the helix. These surfaces
can drive associations of helices, forming a hydrophobic core. B, The
oxaldie enzymatic peptides17 make dual use of this repeating pattern,
creating one hydrophobic, leucine-rich face and one cationic, lysine-rich
face. Spatial clustering of lysines lowers their pKa and provides a surface for
the negatively charged oxaloacetate substrate to bind. L = leucine (grey),
K = lysine (red), A = alanine(white).
Nature chemistry doi: 10.1038/nchem.473
with a hydrophobic Leu/Val-rich face, which binds two 16-residue
peptides that are N and C‑terminal halves of the same sequence.
Binding reduces the entropic costs of chemical ligation between a
C‑terminal thioester leaving group and an N‑terminal cysteine. The
product also serves as a template, resulting in progressive amplifica-
tion of catalytic activity. Although computational methods have not
been applied in the design of these systems, they could potentially
be used to improve catalytic efficiency, by developing sequences
that balance the competing processes of template formation and
product inhibition.
The α‑helix continues to be a useful tool in the rational design
of enzymes without the need for sophisticated computation. The
polar/nonpolar sequence periodicity of the helix serves as a sim-
ple mechanism for promoting multimer associations, and for spa-
tially clustering active-site residues. β‑Hairpins and small sheets
have not been used extensively as a platform for developing small
peptide catalysts, given the increased difficulty of designing folded
β‑structures, which are stabilized by interactions well-separated in
the primary sequence. Significant progress in β‑sheet design may
soon change this23–25. There are limitations to the chemical complex-
ity one can achieve on the face of a single helix. As such, a number of
enzyme designs have made use of more complex protein topologies,
combining multiple helical elements.
Enzyme models with tertiary structure
Valuable progress has been made in the design of catalytic proteins
where active-site and second-shell residues are donated by multi-
ple structural elements. This significantly enhances the potential
for chemical diversity, as well as providing space for binding sites to
improve affinity and specificity. One productive scaffold is the helix–
loop–helix motif, where a short turn connects two amphipathic
α‑helices, which then dimerize into a four-helix bundle26. Because
the two helices are part of the same peptide, they can host a greater
diversity of chemical groups. The Baltzer lab has used this motif to
target a number of reactions involving model RNA-like substrates
(Fig. 2). Functionalizing one face of a helix–loop–helix at four sites
with a zinc-triazacyclononane amino acid resulted in a peptide
capable of catalysing transesterification of 2‑hydroxypropyl‑p-nitro-
phenyl phosphate 380 times faster under saturating conditions27.
The same scaffold has been used to hydrolyse phosphodiester bonds
using histidine as a general acid/base instead of metals28,29.
A three-helix design (helix–loop–helix–loop–helix) with a sin-
gle tryptophan or tyrosine in the hydrophobic core was designed
to measure how the protein environment modulates the stability
of amino acid radicals, as inferred from electrochemical measure-
ments. Amino acid radicals are important in a number of funda-
mental biochemical reactions including the evolution of molecular
oxygen from water by photosystem II and the biosynthesis of DNA
from ribonucleotides by ribonucleotide reductase. The low dielec-
tric environment inside the protein was shown30 to significantly
raise the reduction potential of both Tyr and Trp. The structure of
α3W also uncovered a cation–π interaction between the Trp and
an Arg, which modelling suggested would also raise its reduction
potential31. Although the α3Y/W peptides have not yet been devel-
oped into a catalyst, they showcase an important function of protein
design — to provide minimal systems to help us understand the
complex molecular forces involved in enzyme function.
Binary pattern design of an artificial oxygen-transporter
As discussed above for helichrome, oxalide and others, simple folds
such as helical bundles can be designed non-computationally with
a high probability of success merely by placing polar amino acid
side chains at solvent-exposed positions, and nonpolar amino acids
at core positions, a process termed binary patterning 6. Such design
experiments typically result in molten globules that exhibit a stable
secondary structure but lack a unique three-dimensional structure,
nature chemistry | VOL 2 | JANUARY 2010 | www.nature.com/naturechemistry 17
© 2010 Macmillan Publishers Limited. All rights reserved.
review article
HN1 α3W
Figure 2 | Helical designs with tertiary structure. Using short connecting
loops, multi-helical structures provide the potential for greater chemical
diversity at the active site, a binding surface for defining substrate specificity
and a core for controlling the microenvironment of key catalytic residues.
Two examples are the HN1 ribonuclease29 with an active site of two histidines
and four arginines (model structure), and α3W (ref. 31), a three-helix bundle
that tunes the reduction potential of a tryptophan radical in the bundle core.
most likely because the randomly selected core residues lack ‘knobs
and holes’ type intercalation, allowing these stable elements of sec-
ondary structure to move independently 32–34. Binary patterning cou-
pled with explicitly designed polar interactions buried within the
protein core can fix these elements of secondary structure, lifting
these proteins into unique three-dimensional conformations, either
using internal hydrogen bonding or metal-ligand interactions34,35.
This design approach been exploited in an artificial oxygen
transport protein, HP‑7 (ref. 36). In this protein one of the two lig-
and histidines of a bis-histidine haem binding site in a binary pat-
terned four-helix bundle scaffold was destabilized by the addition
of core glutamic acid residues on the same helix. This intentional
violation of the rules of binary patterning results in the stabiliza-
tion of an alternative conformation wherein the ligand histidine
detaches and the core glutamate residues rotate out into solution,
opening a haem iron coordination site for oxygen binding (Fig. 3).
This mechanism is similar to that observed for other hexacoordi-
nate hemoglobins such as cytoglobin, neuroglobin or leghemo-
globin37, although the structure and sequence of HP‑7 is unrelated
to any natural oxygen carrier.
Measurement of the kinetic and thermodynamic constants for
oxygen and carbon monoxide binding by HP‑7 demonstrates that
it performs equivalently to natural globins, with the unprecedented
O2
Haem
Figure 3 | Stepwise oxygen binding to a binary-patterned four-helix
bundle. In the HP‑7 maquette36, haem binding requires rotation of α‑helices
to present histidines (green) in the proper geometry. This forces the
unfavourable burial of glutamates (red triangles) inside the nonpolar protein
core. Release of one of the axial histidines allows the glutamates to interact
with solvent and provides an open coordination site on the haem for oxygen
binding. Figure reproduced with permission from ref. 36, © 2009 NPG.
review article
exception that HP‑7 binds molecular oxygen more tightly than car-
bon monoxide. This work not only demonstrates that an intuition-
led approach like the modified binary-patterning method described
here can lead to sophisticated biological function, it presages pro-
tein design’s ability to exceed the capabilities of the proteins found
in nature.
Computational design of a de novo enzyme
Designing effective enzymes requires the ability to design structure.
This is an area where computational methods have proved most
useful. Fully automated designs of sequences to achieve target folds
have been demonstrated multiple times over the past decade, from
small proteins such as a zinc-finger-like structure that folds without
metal38 to Top7 (ref. 39), a novel mixed α/β fold with no natural
counterpart. Although the fully automated design of an enzyme
from the ground up has yet to be accomplished, recent work on
computationally designed metalloenzymes have made significant
advances in this area.
Computational design had an appreciable role in the DF series of
proteins (due ferro, Italian for ‘two iron’). These were inspired by a
class of natural oxygen activating enzymes with dinuclear iron clus-
ters40. A retrostructural analysis of metalloenzymes such as the R2
subunit of ribonucleotide reductase and methane monooxygenase
revealed a simple underlying D2 symmetry of the protein backbone,
an antiparallel four-helix bundle, which reflected the symmetry of
the active-site metals and substrates41 (Fig. 4). This made it possible
to generate a scaffold de novo using an ideal α‑helix and symmetry
operations42. Key metal ligands (called keystone interactions) and
second-shell interactions were placed on the scaffold. The protein
was designed using a combination of manual and automated mod-
elling. One of the computational tools used was ROC (repacking
of cores), a program that optimizes a design by evaluating struc-
ture/sequence candidates on van der Waals energies of side-chain
packing 43. The final model was a four-helix bundle composed of a
homodimer of helix–loop–helix chains, much like those of Baltzer26.
The atomic structure of the design was solved by X‑ray crystallog-
raphy in the presence of zinc. Comparison of the structure and the
model demonstrated that many of the design elements were suc-
cessfully implemented. DF1 provided a clear link between sequence,
structure and function, making it a rich platform for understanding
how this class of proteins functions.
The structure of DF1 suggested that any potential catalytic
activity was hampered by the presence of two leucines at the metal
site. These were mutated to alanine in DF2, providing a substrate
channel capable of binding small aromatic molecules44. On adding
4‑aminophenol (4-AP), it was shown that DF2 could catalyse two-
electron oxidation to benzoquinone monoimine through a diferric
Glu
His
Glu
Ribonucleotide Metal site DF-1
reductase R2 symmetry
Figure 4 | Retrostructural analysis and design of a dinuclear
metalloprotein. The DF series of designed metalloenzymes were built from
structural analysis of dinuclear metal sites in proteins such as ribonucleotide
reductase41. The two metals, two histidines and four glutamates that formed
the active site could be described by two half-sites related by a C2 symmetry
axis. The same symmetry was found locally in the natural metalloenzymes
and was used in the de novo design of a helix–loop–helix dimer, DF‑1.
18 nature chemistry | VOL 2 | JANUARY 2010 | www.nature.com/naturechemistry
© 2010 Macmillan Publishers Limited. All rights reserved.
Nature chemistry doi: 10.1038/nchem.473
intermediate in the presence of oxygen; multiple turnovers and
thousandfold rate enhancements over the uncatalysed reaction were
observed45. Using an electron-rich phenol such as 4‑AP provided
a facile substrate for establishing structural features necessary for
binding and catalysis. Additional optimization through combinato-
rial libraries or directed evolution could be used to develop more
powerful metalloenzymes.
The DF series has also proved to be a useful tool in understanding
how natural metalloenzymes may fold and function. One important
feature of natural enzymes recapitulated by DF is preorganization.
Preorganization of active-site residues improves catalytic activity by
reducing the entropic cost of forming the enzyme–substrate com-
plex. It also allows the enzyme to dictate the configuration of the
complex, which may be important for targeting transition states
and high-energy intermediates. In the absence of metal, the fold
was highly similar and key metal binding residues were constrained
close to the active site46. Another feature that distinguishes enzymes
from small-molecule catalysts is their ability to couple reactivity to
protein motion, such as allosteric conformational changes that tog-
gle an enzyme between active and inactive states. In high-resolution
structures of DF1 bound to manganese, two coordination envi-
ronments were found in the asymmetric unit: one where a solvent
molecule bridges the two metals, and a second where two solvent
molecules are bound trans to protein ligands47,48. This was coupled
with a shift between the two helix–loop–helix motifs, suggesting
that a large-scale sliding motion could tune active-site reactivity.
The DF series has also helped further general computational
methods in enzyme design and simulation. In work to create a sin-
gle chain protein, DFsc, it was found that correctly modelling the
turn residues between helices could significantly improve design
stability. This led to an in-depth characterization of helix–turn–helix
structures in the Protein Data Bank (PDB), identifying key turn
motifs that correlated to specific geometries of helix–helix pairs. A
de novo computational simulation of charge-pair interactions on the
surface of DF was used to design sequences that formed a 2A:2B
heterotetramer between four separate helices49. It was even possible
to build an A:B:2C heterotetramer from three peptides using sur-
face electrostatics50. This will facilitate combinatorial approaches
to develop better enzymes by mixing libraries of peptides and
screening for activity. High-resolution active-site design has been
initiated with DF1, subjecting a chemically simplified metal site to
Car–Parrinello molecular dynamics simulations51. Snapshots from
these simulations correspond well to high-resolution structures of
DF1. Simulation methods could be used to generate structures of
transition states and high-energy intermediates in catalysis. These
could then be included as constraints in the redesign of DF1 for
novel catalytic activity.
The retrostructural analysis strategy has also been applied to the
computational design of a β‑sheet metalloprotein and a four-helix
bundle that binds arrays of non-natural porphyrin cofactors52,53. Key
features such as focusing on keystone interactions, binding-site pre-
organization by second-shell ligands, and mirroring of protein fold
and active-site symmetry have been implemented in these systems.
Integration of computation and chemical intuition in the de  novo
design of proteins will further our understanding of the basic rela-
tionship between sequence, structure and reactivity.
Computational de novo active-site design
Although the concurrent design of structure and catalysis prom-
ises to broadly expand the scope of artificial enzymes, this area is
still in its infancy. Even state-of-the-art designs such as DF still
depend heavily on biological motifs and helical topologies. Thus, a
number of labs have pursued the more tractable target of designing
novel enzymatic functionality into existing protein scaffolds. Two
major challenges in de  novo active-site design are (1) identifying
optimal locations on a protein to introduce the active-site residues
Nature chemistry doi: 10.1038/nchem.473
and (2) modelling the active site with sufficient accuracy to enable
appropriate reactivity. These issues represent a long-standing con-
flict in protein design between speed and accuracy. Approximations
in energy calculations and coarse-grained sampling methods
may speed simulations, and allow broader sampling of sequence/
structure combinations. However, the trade-off with accuracy may
reduce the ability to discriminate between successful and unsuc-
cessful designs.
Software such as METAL SEARCH54–56 and DEZYMER57 suc-
cessfully introduced metal-binding sites into novel locations on pro-
tein surfaces. Starting with a high-resolution structure of the target
protein, sites in the backbone were selected based on their capacity
to present metal-binding residues presenting thiol, carboxylate or
imidazole ligands. These were then modelled onto the structure in
various rotameric configurations to determine whether the coordi-
nation geometry of the metal was satisfied.
Despite the comparatively large energy of metal–ligand bond
formation relative to other intramolecular forces in proteins, accu-
rate design of metalloproteins presents a number of challenges, par-
ticularly with regards to specificity. For example, DEZYMER was
used to convert the substrate binding region of maltose binding
protein (MBP) into a zinc-binding site. MBP has two large domains
connected by a flexible hinge region, allowing a cavity between the
two domains to open and close. Maltose binds to this cavity and sta-
bilizes the closed state (Fig. 5). Marvin and Hellinga systematically
modelled groups of amino acids in the domain interface as potential
zinc ligands, until a set of four amino acids were identified, two from
each domain, which could potentially bind zinc when MBP was in
the closed conformation58. The best design bound zinc with micro-
molar affinity. Subsequent biophysical and structural characteriza-
tion revealed an unanticipated mode of zinc binding that involved
the open state59. Two histidines of the four designed amino acid
substitutions were shown to bind the zinc. A nearby aspartate car-
boxylate side-chain was found within four ångströms of the metal,
too far to form direct, strong ligand–metal bonds. However, muta-
tion of this Asp to Ala abolished affinity for zinc, indicating some
role in binding. Thus a key challenge in metalloprotein design is to
understand the role of second-shell and potentially weak first-shell
interactions in affinity and specificity. The large-scale conforma-
tional transitions in MBP between open and closed states high-
lights another challenge — that of negative design. By optimizing
side-chain metal–ligand geometries, programs seek to maximize
the stability of a target state, usually using a crystal structure as a
starting point. Negative design seeks to account for competing, off-
target conformations and either explicitly or implicitly destabilize
them. This is extremely difficult without a detailed knowledge of the
structure of conformational microstates. Optimization of sequence
over an ensemble of target and off-target protein conformations also
exacerbates the already formidable number of states to be sampled.
This is currently a very active area in protein design.
A series of His3Fe sites were introduced to thioredoxin in vari-
ous environments classified as grooves, shallow pockets and a deep
pocket, allowing the effect of the protein microenvironment on
‘nascent’ enzymatic activity to be studied60,61. Superoxide dismutase
activity, the conversion of superoxide radical into molecular oxygen
and hydrogen peroxide, correlated with local electrostatic interac-
tions, where a net positive charge at the binding site was hypoth-
esized to attract the O2– species. Further structural characterization
of these designs could be very informative, as the previous MBP
example emphasizes. Where possible, an atomic-resolution struc-
ture of a de novo functional protein is an important step in under-
standing how design elements correlate to mechanism.
A similar approach was used to build a non-metal proto-enzyme
site into a thioredoxin scaffold that catalysed the hydrolysis of
paranitrophenol acetate (PNPA) into PNP and acetate62. This was
accomplished by a histidine nucleophile that formed a high-energy
nature chemistry | VOL 2 | JANUARY 2010 | www.nature.com/naturechemistry 19
© 2010 Macmillan Publishers Limited. All rights reserved.
review article
a b
E155
E340
D65
H63
H66
Figure 5 | Design versus structure of a Zn2+ binding MBP. a, Using the
computer program DEZYMER57, four substitutions were designed into closed
form of the maltose binding protein (MBP), which conferred affinity for zinc.
Mutations (in yellow) were in the maltose binding site (white spheres).
b, Crystal structures of the MBP variant demonstrated binding was in fact to
a conformation closer to the open form, only involving half of the residues
and an additional aspartate ligand (green). A = alanine, R = arginine,
Y = tyrosine, W = tryptophan, E = glutamate, D = aspartate, H = histidine.
transition state with PNPA. To computationally model this reaction,
a composite side chain composed of the histidine covalently linked
to PNPA was introduced and conformationally sampled around
accessible bond rotations. Adjacent amino acids to the site of the
His-PNPA were allowed to mutate to alanine to facilitate substrate
binding and recognition. The conformations of His-PNPA and sur-
rounding side chains were optimized using Dead End Elimination,
a powerful algorithm that significantly reduces the combinatorial
complexity of multi-site optimizations by eliminating pairwise
states that are provably incompatible with the global energy mini-
mum63. The top two scoring candidates, PZD1 and 2 were synthe-
sized. PZD2 demonstrated significant rate enhancements over the
uncatalysed reaction and saturation kinetics with increasing sub-
strate concentration.
Although the thioredoxin-derived metalloenzymes and PDZ2
had kinetic parameters well below those of natural enzymes and
even catalytic antibodies, they were key first steps in developing
computational methods for enzyme design. Important advances
in the efficiency of computational methods such as Dead End
Elimination are proving crucial in making these design problems
tractable64. Although initial extension of these computational meth-
ods to the design of a triose phophate isomerase turned out to be
unsuccessful, many important ideas put forth in these studies were
incorporated into the recent, successful design of chemically ambi-
tious artificial enzymes.
In parallel with computational advances, active-site designs con-
tinue to progress using rational, intuition-based strategies. Esterase
activity was introduced to human carbonic anhydrase (HCAIII)
through both protein and substrate engineering 65. The affinity of
HCAIII for benzenesulfonamide-containing molecules was used to
model a substrate such that the scissile bond was positioned within
a cleft in the protein. Grafting a His dyad from previous de  novo
helix–loop–helix designs resulted in an HCAIII variant with
enhanced esterase activity over wild-type.
The ROSETTA enzymes
ROSETTA is a suite of computational tools developed in the labo-
ratory of David Baker for protein-structure prediction, protein–
protein complex prediction and protein design. One of the key
innovations in ROSETTA is its use of high-resolution structures
in the PDB as a ‘parts list’. Small fragments around ten residues in
length are assembled into larger molecules, drastically reducing the
conformational degrees of freedom to be sampled. This approach
review article Nature chemistry doi: 10.1038/nchem.473
a
O

Motif III O
Asp

H
Hydrophobic
pocket N

O OH N His O O
Lys H +
NH O

O O

b Theozyme c Active site ensemble: 1018 states

His

Asp

Lys

d Scaffold set e Matches

Jelly roll TIM barrel

Figure 6 | Assembly line for the ROSETTA Enzymes. a, Sketch of a reaction motif, outlining key intermediates, general acid/base ligands and strategies
for modulating catalytic residue pKas. b, QM calculations are used to optimize the geometry of a transition-state model including truncated active-site
residues. c, Elaboration of catalytic residue rotamers creates an ensemble of active sites. d, These sites are matched to complementary surfaces on a
family of target protein scaffolds. e, Promising designs are synthesized and characterized for activity. Figure reproduced from ref. 111, © 2008 NPG.

rests on the assumption that structure and stability information is
implicitly encoded locally within each fragment 34. The global stabil-
ity of a design is evaluated based on a scoring potential that com-
bines physics-based energy terms such as van der Waals packing
and knowledge-based energy terms derived from statistical analysis
of amino acid interactions with the PDB66. Recently, ROSETTA was
used to develop artificial enzymes that catalysed a retro-aldol reac-
tion (Fig. 6) and a Kemp elimination67,68. These designs were impres-
sive in the extent to which the relationship between structure and
reactivity was modelled and characterized.
In the retro-aldolase, the goal was to break a carbon–carbon bond
in a non-natural substrate, 4‑hydroxy‑4-(6-methoxy‑2-napthyl)‑2-
butanone69. The intended reaction was significantly more complex
than previous designs, involving multiple transition states and
intermediates to cleave the substrate and regenerate the active site.
The reaction proceeded through an imine intermediate involving
a lysine as a Schiff base, similar to the oxaldie decarboxylation of
oxaloacetate. In oxaldie, the lysine nucleophile was stabilized through
electrostatic interactions with other charged side chains or the helix
macrodipole. The same mechanism was used in the first of four reac-
tion motifs attempted by placing a second lysine in the vicinity of
the first. The other three used a hydrophobic pocket to lower the
pKa of the lysine. A general acid/base was included to trigger cleav-
age of the carbon–carbon bond. Each motif used a different base:
I, a Lys/Asp dyad; II, tyrosine; III, a His/Asp; and IV, a water mol-
ecule. Attempting designs based on several reaction motifs not only
increases the chance of a successful outcome, but also demonstrates
how design can be used to test various hypotheses for catalysis.

20 nature chemistry | VOL 2 | JANUARY 2010 | www.nature.com/naturechemistry

© 2010 Macmillan Publishers Limited. All rights reserved.
Nature chemistry doi: 10.1038/nchem.473
As previously discussed, two challenges in computational
enzyme design are accurate modelling of active-site interactions
and sufficient sampling of candidate backbone templates on protein
scaffolds. To meet the first challenge, quantum mechanics calcu-
lations were performed on a minimal chemical representation of
the protein–substrate complex 70. To simplify active-site construc-
tion, a composite transition state was made that combined optimal
geometries of the carbinolamine alcohol and the bond-breaking
state. The remainder of the active-site side-chains were then built
and sampled for rotameric degrees of freedom. After all active-site
and second-shell residues were modelled, a total of anywhere from
1013 to 1018 potential sequence/structure combinations were gener-
ated, depending on the reaction motif considered.
At this point in the design, the researchers had built a huge
ensemble of disembodied active sites. The next challenge was to
compare these active sites to a set of potential scaffolds in seventy
protein structures. These structures represented a diverse set of
protein folds including triose phosphate isomerase (TIM) barrels,
jelly rolls, β‑propellors, lipocalins and periplasmic binding proteins.
To model 1018 active sites onto all possible combinations of back-
bone positions would be computationally intractable. An algorithm
called RosettaMatch made use of geometric hashing to reduce the
problem to one that scaled linearly with the number of states71,72.
(For a very readable review of geometric hashing, we recommend
ref. 73.) Still, this was an immense calculation, and other resources
such as distributed computing over thousands of personal comput-
ers volunteered through the Rosetta@Home project made this pos-
sible. After the first stage of matching, the number of designs was
around 180,000. This was further reduced by computational rede-
sign of adjacent amino acids surrounding the active site, in order to
maintain structural integrity of the overall protein fold.
After an extensive computational vetting process, searching
through four reaction motifs, a billion billion active-site configu-
rations and a diverse set of scaffolds, seventy proteins were syn-
thesized. Each design contained anywhere from eight to twenty
mutations relative to the wild-type protein. Impressively, nearly
half showed some aldolase activity. Interestingly, the successful
enzymes were not equally distributed over mechanism or fold. Only
motifs III and IV, which used histidine or water as the general acid/
base, were successful, suggesting the other mechanisms were either
chemically unfeasible or difficult to design. Similarly, only the jelly-
roll and TIM barrel were productive, indicating these folds may
have intrinsic geometric properties favouring the design of catalytic
sites (the capacity for a fold to accommodate multiple sequences
is referred to as designability, and quantifying this parameter has
been attempted for simple systems)74,75. Rate enhancements of up
to 104 were achieved. Structures of designs solved by X‑ray crystal-
lography showed the atomic accuracy of the computational models,
an essential feature for furthering rational design of these enzymes.
Designs were verified by mutagenesis of active-site residues to ablate
catalysis. Careful analysis verified saturation kinetics. Although
these designs fall short of retro-aldolase activity seen in catalytic
antibodies69,76, they remove a number of constraints on the design of
artificial enzymes, opening the possibility for reactivity on a broad
spectrum of protein scaffolds.
This approach can also be combined with in  vitro evolution to
improve catalytic efficiency, as was shown with a set of Kemp elimi-
nation enzymes also designed with the ROSETTA platform68. The
computational approach to designing Kemp elimination enzymes
was very similar to the retro-aldolase. Kemp elimination is a model
reaction for proton abstraction from a carbon. Successful designs
were again found in TIM-barrel folds, with rate enhancements of
up to 105, saturation kinetics in certain cases and greater than seven
turnovers. In vitro evolution of one of the designs, KE07, by cycles of
mutagenesis and activity screening resulted in a 200-fold improve-
ment in kcat/Km. Residues involved in catalysis were unchanged;
nature chemistry | VOL 2 | JANUARY 2010 | www.nature.com/naturechemistry 21
© 2010 Macmillan Publishers Limited. All rights reserved.
review article
rather, most mutations were found in amino acids adjacent to the
active site. High-resolution crystal structures of KE07 were super-
imposable on the computational model, allowing the researchers to
develop specific hypotheses regarding the mechanism of improved
enzymatic function by evolution. This highlights the potential advan-
tage of combining rational design with directed evolution.
As with retro-aldolase designs, KE07 only demonstrates kinetic
parameters equivalent to or below that of catalytic antibodies77, or
even non-specific reaction rates with ‘off the shelf ’ proteins such
as bovine and human serum albumins78. The real success of the
ROSETTA enzymes is not in the computational design of catalysts
of practical value, a goal that has still to be achieved by any group.
Rather, they are among the earliest demonstrations of the ability to
harness the three-dimensional protein fold to stabilize energetically
unfavourable active sites79, for example by locating a general base in
a hydrophobic microenvironment. This is an important component
of enzyme design.
A two-pronged approach combining laboratory evolution with
structure-based computation may increase the likelihood of design-
ing synthetic enzymes with activities approaching those of natural
counterparts. One strategy is to reduce the number of sequences
to be experimentally characterized. This has been approached in
multiple ways: identifying positions in a protein that can tolerate
mutations80, identifying protein domain boundaries in order to gen-
erate libraries of permuted chimeric species81 and predicting amino
acid substitutions that preserve function while maximizing chemi-
cal diversity of sampled sequences82. The Tidor laboratory took an
inverse approach, using computational sequence optimization to
enhance antibody binding after affinity maturation against two tar-
gets (lysozyme and human epidermal growth factor receptor)83.
Moving enzyme design out of the active site
KE07 models a potentially powerful strategy of combining computa-
tional design and laboratory evolution towards novel enzymes. Below,
we highlight other areas where rational methods may introduce new
functionality that would be difficult using laboratory evolution alone.
The enzymes designed so far have each focused on the creation of
a transition-state-stabilizing active site as the basis for their catalytic
function. Thus, they necessarily have the same limitations in scope as
has been noted for catalytic antibodies elicited with transition-state
analogues76, in particular their modest catalytic rate accelerations
that pale in comparison to the values of up to 1023 observed in natural
enzymes84. This is at least in part due to the fact that catalysts that bind
to transition states too tightly suffer from product inhibition, setting
a limit on the maximal rate acceleration possible using this method
alone85. For rationally designed enzymes to surpass these limitations,
additional strategies, many derived from natural enzyme mecha-
nism, must be employed86. Although these strategies incur an addi-
tional level of complexity in rational design, they are also responsible
for much of the extra catalytic power observed in protein catalysts.
Some of these further mechanistic enhancements may offer an entry-
way into catalytic complexity that is not only impossible in catalytic
antibodies, but also not accessible to directed evolution approaches.
Furthermore, it seems likely that many of these strategies are them-
selves open to implementation using chemical intuition.
Multiple substrates. Perhaps the simplest mechanism by which
enzymes accelerate chemical reactions is by using binding energy to
compensate for the entropy loss incurred in bimolecular reactions87.
Merely holding two substrates in proximity to each other at the cor-
rect orientation for a productive reaction can engender a 108-fold
rate acceleration in the absence of any transition-state-stabilizing
protein–substrate interactions. Such a strategy proved successful in
the self-replicating peptide system21. This, coupled with transition–
state-stabilization, could elevate rate enhancements to levels similar
to those observed in natural enzymes. Designing a single active site
review article
Figure 7 | Millisecond timescale motions in adenylate cyclase. Adenylate
cyclase maintains intracellular concentrations of adenylate nucleotides
by converting an ATP and an AMP into two ADP molecules. Binding and
catalysis requires a significant conformational rearrangement93,94. The two
domains of adenylate cyclase are shown in red and grey: left, the unbound
form; right, bound to a dinucleotide analogue shown in blue.
that will bind, orient and activate two substrates simultaneously is,
however, a more difficult problem. It seems likely that simpler bi-
substrate mechanisms, either an ordered mechanism with a cova-
lent intermediate, or a single protein with two active sites coupled
by the channelling of an intermediate, would be more accessible to
current design technology 3.
Timing in multiple substrate reactions. An important facet of
the mechanism of many of nature’s more complicated catalysts is
the temporal control of substrate binding and transfer events. One
merely has to look at the exquisite engineering apparent in the
multiple electron and proton transfers in photosynthesis, respira-
tion and nitrogen fixation to see the advantages conferred by the
ability to control the timing and energetics of the intermediates in
multiple-substrate reactions88. As these reaction mechanisms would
require the simultaneous optimization of several different catalytic
events at several different sites, it is unlikely that such would arise
from laboratory timescale evolution. A more likely situation is one
where an initial enzymatic scaffold is designed explicitly and then
the kinetics and thermodynamics of the intermediates are further
optimized using directed evolution.
Small-scale enzyme motions. The analysis of hydrogen tunnelling
in enzymes has established unequivocally that protein dynamics
can play a large part in rates of enzymatic catalysis89. Computational
analysis of enzyme–substrate complexes using molecular mechanics
has estimated the role of the dynamics of ‘near attack complexes’ in
catalytic function in the absence of atomic tunnelling to be as high
as 103 (ref. 90). However, these nanosecond timescale motions are
an intrinsic property of all biopolymers. It is not clear to what extent
choreographed fast dynamics promotes catalysis in different natural
enzymes. A recent report of the dynamic analysis of each interme-
diate state in the catalytic mechanism of dihydrofolate reductase91
demonstrated that in each kinetic intermediate, the protein accessed
only the conformations present in the current state and the states
immediately before and after. These findings indicate that for at least
some enzymes such dynamics are considerably choreographed.
Because the origins and even the consequences of these motions
are not well understood, this contribution to catalysis will be dif-
ficult to reproduce. As the optimization brought about by directed
evolution techniques is often a result of mutations distal to the active
site that may impact protein motions, it seems that this method is
currently the best approach for increasing catalytic efficiency with
22 nature chemistry | VOL 2 | JANUARY 2010 | www.nature.com/naturechemistry
© 2010 Macmillan Publishers Limited. All rights reserved.
Nature chemistry doi: 10.1038/nchem.473
small-scale dynamics. Designed enzymes offer a uniquely adaptable
scaffold on which to test our ideas about such dynamic motions,
and artificial proteins will no doubt prove to be central in the devel-
opment of our understanding of enzyme dynamics.
Large-scale enzyme motions. Multi-ångström, millisecond times-
cale protein motions are a critical component of many natural enzyme
mechanisms92. One well-characterized example is the Ω loop govern-
ing the function of adenylate kinase93,94, where this loop has an open
conformation, competent for substrate binding and product release,
and a closed conformation, competent for catalysis (see Fig. 7). This
hinge-opening motion on a ‘lid’ over the active site is a common motif
in protein function, particularly the TIM-barrel family of enzymes95.
There are even larger motions, involving entire protein domains, used
by complex multidomain enzymes — the ‘escapement’ mechanisms
that have been well characterized in the cytochromes bc1 and the fla-
vocytochrome P450 BM396 are simple large-scale motions. Intuitive
approaches can be envisaged for incorporating large-scale motions
into a protein design, such as the introduction of flexible loops. Such
features are unlikely to result directly from laboratory timescale evo-
lution approaches without some degree of initial design.
The helical rotation discussed above in the mechanism of the
artificial neuroglobin HP‑7, is exemplary of the types of problems
de novo enzyme design can target relative to catalytic antibody for-
mation or directed evolution. Antibodies are relatively inflexible
and evolutionary methods are unlikely to generate multiple con-
formations simultaneously. As such conformational switching is an
essential component of the function of many protein catalysts; these
motions are best incorporated at the design level.
Allostery. Cooperative phenomena are fundamental in many bio-
logical functions such as oxygen transport, metabolic and transcrip-
tional regulation97. The ability to incorporate allosteric regulation
into artificial enzymes will enable the creation of medicinally useful
in vivo catalysts that can be regulated either by metabolites or using
exogenous small-molecule effectors. Such behaviour is in most
cases a more complicated version of the large-scale conformational
switching described above, only in this case the large-scale protein
motion is actuated by the binding of an effector molecule.
Keeping energetic intermediates from the cellular environment.
Anyone who has observed a bioinorganic chemist at work in a dry
glovebox can appreciate the fragility of metalloenzyme active sites.
Such enzymes employ reactive intermediates that must be screened
from water, oxygen and reactive species in the cellular environment
such as glutathione and superoxide. In fact, the failure of many ini-
tial attempts at metalloenzyme design can be attributed to the desire
to make these model proteins as small as possible98. Larger proteins
have sufficiently sized hydrophobic cores that they can completely
encapsulate these intermediates in a non-reactive environment,
screening them from solution and lengthening their lifetimes.
Catalytic foldamers
Despite the intensive focus on protein enzymes, the first catalytic
biomolecules may have been based on RNA rather than amino
acids99. Nucleotides can carry out metal-assisted reactions, act as
general acid–bases and function to orient substrates and isolate
them from solvent, much like the most sophisticated catalytic pro-
teins100. Catalytic activity has also been found in certain bacterial
carbohydrates101. Evidently, proteins do not have a monopoly on
biological catalysis. Although the successful design of a new pro-
tein is a rewarding experience in itself, often real advances are in
our understanding of the basic molecular forces that guide struc-
ture and function. Given that catalysis is not limited to proteins,
it is important for us to ask whether insights gained from current
de  novo proteins are idiosyncratic to proteins, or whether we are
Nature chemistry doi: 10.1038/nchem.473
learning broader lessons about molecular design. This is the goal
of ‘foldamer’ research, the development of novel polymeric systems
that fold into unique, three-dimensional structures102,103.
Towards designing a functional foldamer, it is important to
remember the lessons of oxaldie and other catalytic peptides — a
firm grasp of molecular structure is an important prerequisite to
introducing function. Computational studies of foldamers are still
in their infancy. Several groups have used simulation approaches to
study the conformational space available to peptidomimetics incor-
porating d‑amino acids104,105 or β‑amino acids106,107, as well as com-
pletely non-biological foldamers such as m‑phenylene ethylenes
(mPE)108. These studies establish non-natural scaffolds that can then
be functionalized to facilitate reactivity. The mPEs are promising
owing to their capacity to form helical cavities that can be func-
tionalized with catalytic residues and serve to isolate substrate from
solvent 109. This is an exciting new direction for molecular design.
Although the Wright brothers did not use a computer to build
the first airplane, the aerospace industry has since greatly benefitted
from sophisticated simulation and design software. Clearly, both the
chemist and the computer will have important roles in the future of
artificial enzymes. Design by chemical intuition tests our understand-
ing of basic rules, that is, binary patterning, molecular forces, metal
coordination and catalytic mechanisms. These rules are then codified
to allow the computational design of increasingly complex systems.
Designer enzymes are a useful test of our understanding of how pro-
teins fold and function. They also provide a rational path towards
inexpensive, non-toxic catalysts that perform novel chemistry.
References
1. Davie, E. A. C., Mennen, S. M., Xu, Y. & Miller, S. J. Asymmetric catalysis
mediated by synthetic peptides. Chem. Rev. 107, 5759–5812 (2007).
2. List, B. Proline-catalyzed asymmetric reactions. Tetrahedron
58, 5573–5590 (2002).
3. Koder, R. L. & Dutton, P. L. Intelligent design: the de novo engineering of
proteins with specified functions. Dalton Trans. 25, 3045–3051 (2006).
4. Lim, V. in Protein Folding, 28th Conference of the German Biochemical Society
(ed. Jaenicke, R.) 149–166 (Elsevier, 1979).
5. Crick, F. H. C. The packing of alpha-helices: simple coiled coils. Acta Crystallogr.
6, 689–697 (1953).
6. Kamtekar, S., Schiffer, J. M., Xiong, H., Babik, J. M. & Hecht, M. H. Protein
design by binary patterning of polar and nonpolar amino acids. Science
262, 1680–1685 (1993).
7. Lau, S. Y. M., Taneja, A. K. & Hodges, R. S. Synthesis of a model
protein of defined secondary and quaternary structure. J. Biol. Chem.
259, 13253–13261 (1984).
8. DeGrado, W. F. & Lear, J. D. Induction of peptide conformation at apolar/
water interfaces. 1. a study with model peptides of defined hydrophobic
periodicity. J. Am. Chem. Soc. 107, 7684–7689 (1985).
9. Bryson, J. W. et al. Protein design: a hierarchic approach. Science
270, 935–941 (1995).
10. Handel, T. M., Williams, S. A. & DeGrado, W. F. Metal ion-
dependent modulation of the dynamics of a designed protein. Science
261, 879–885 (1993).
11. Lovejoy, B. et al. Crystal structure of a synthetic triple-stranded alpha-helical
bundle. Science 259, 1288–1293 (1993).
12. Harbury, P. B., Plecs, J. J., Tidor, B., Alber, T. & Kim, P. S. High-resolution
protein design with backbone freedom. Science 282, 1462–1467 (1998).
13. Sasaki, T. & Kaiser, E. T. Helichrome: synthesis and enzymatic activity of a
designed hemeprotein. J. Am. Chem. Soc. 111, 380–381 (1989).
14. Barbier, B. & Brack, A. Basic polypeptides accelerate the hydrolysis of
ribonucleic acids. J. Am. Chem. Soc. 110, 6880–6882 (1988).
15. Barbier, B. & Brack, A. Conformation-controlled hydrolysis of
polyribonucleotides by sequential basic polypeptides. J. Am. Chem. Soc.
114, 3511–3515 (1992).
16. Brack, A. & Spach, G. Multiconformational synthetic polypeptides. J. Am.
Chem. Soc. 103, 6319–6323 (1981).
17. Johnsson, K., Allemann, R. K., Widmer, H. & Benner, S. A. Synthesis,
structure and activity of artificial, rationally designed catalytic polypeptides.
Nature 365, 530–532 (1993).
18. Taylor, S. E., Rutherford, T. J. & Allemann, R. K. Design, synthesis and
characterisation of a peptide with oxaloacetate decarboxylase activity. Bioorg.
Med. Chem. Lett. 11, 2631–2635 (2001).
nature chemistry | VOL 2 | JANUARY 2010 | www.nature.com/naturechemistry 23
© 2010 Macmillan Publishers Limited. All rights reserved.
review article
19. Taylor, S. E., Rutherford, T. J. & Allemann, R. K. Design of a folded,
conformationally stable oxaloacetate decarboxylase. J. Chem. Soc. Perkins
Trans. 2, 751–755 (2002).
20. Nicoll, A. J. & Allemann, R. K. Nucleophilic and general acid catalysis at
physiological pH by a designed miniature esterase. Org. Biomol. Chem.
2, 2175–2180 (2004).
21. Lee, D. H., Severin, K., Yokobayashi, Y. & Ghadiri, M. R. Emergence of
symbiosis in peptide self-replication through a hypercyclic network. Nature
390, 591–594 (1997).
22. Saghatelian, A., Yokobayashi, Y., Soltani, K. & Ghadiri, M. R. A chiroselective
peptide replicator. Nature 409, 797–801 (2001).
23. Butterfield, S. M., Cooper, W. J. & Waters, M. L. Minimalist protein design: a
beta-hairpin peptide that binds ssDNA. J. Am. Chem. Soc. 127, 24–25 (2005).
24. Butterfield, S. M., Goodman, C. M., Rotello, V. M. & Waters, M. L. A peptide
flavoprotein mimic: flavin recognition and redox potential modulation in
water by a designed beta hairpin. Angew. Chem. Int. Ed. 43, 724–727 (2004).
25. Hughes, R. M. & Waters, M. L. Model systems for beta-hairpins and beta-
sheets. Curr. Opin. Struct. Biol. 16, 514–524 (2006).
26. Olofsson, S. & Baltzer, L. Structure and dynamics of a designed helix-loop-
helix dimer in dilute aqueous trifluoroethanol solution. A strategy for NMR
spectroscopic structure determination of molten globules in the rational
design of native-like proteins. Fold. Des. 1, 347–356 (1996).
27. Rossi, P., Tecilla, P., Baltzer, L. & Scrimin, P. De novo metallonucleases based
on helix‑loop‑helix motifs. Chem. Eur. J. 10, 4163–4170 (2004).
28. Razkin, J., Lindgren, J., Nilsson, H. & Baltzer, L. Enhanced complexity and
catalytic efficiency in the hydrolysis of phosphate diesters by rationally
designed helix‑loop‑helix motifs. ChemBioChem 9, 1975–1984 (2008).
29. Razkin, J., Nilsson, H. & Baltzer, L. Catalysis of the cleavage of uridine 3’‑2,2,
2-trichloroethylphosphate by a designed helix‑loop‑helix motif peptide. J. Am.
Chem. Soc. 129, 14752–14758 (2007).
30. Tommos, C., Skalicky, J. J., Pilloud, D. L., Wand, A. J. & Dutton, P. L. De novo
proteins as models of radical enzymes. Biochemistry 38, 9495–9507 (1999).
31. Dai, Q. H. et al. Structure of a de novo designed protein model of radical
enzymes. J. Am. Chem. Soc. 124, 10952–19053 (2002).
32. Gibney, B. R., Rabanal, F., Skalicky, J. J., Wand, A. J. & Dutton, P. L. Design of a
unique protein scaffold for maquettes. J. Am. Chem. Soc. 119, 2323–2324 (1997).
33. Gibney, B. R., Rabanal, F., Skalicky, J. J., Wand, A. J. & Dutton, P. L. Iterative
protein redesign. J. Am. Chem. Soc. 121, 4952–4960 (1999).
34. Simons, K. T., Kooperberg, C., Huang, E. & Baker, D. Assembly of
protein tertiary structures from fragments with similar local sequences
using simulated annealing and bayesian scoring functions. J. Mol. Biol.
268, 209–225 (1997).
35. Koder, R. L. et al. Native-like structure in designed four helix bundles driven
by buried polar interactions. J. Am. Chem. Soc. 128, 14450–14451 (2006).
36. Koder, R. L. et al. Design and engineering of an O2 transport protein. Nature
458, 305–309 (2009).
37. Kundu, S., Trent, J. T. & Hargrove, M. S. Plants, humans and hemoglobins.
Trends Plant Sci. 8, 387–393 (2003).
38. Dahiyat, B. I. & Mayo, S. L. De novo protein design: fully automated sequence
selection. Science 278, 82–87 (1997).
39. Kuhlman, B. et al. Design of a novel globular protein fold with atomic-level
accuracy. Science 302, 1364–1368 (2003).
40. Wallar, B. J. & Lipscomb, J. D. Dioxygen activation by enzymes containing
binuclear non-heme iron clusters. Chem. Rev. 96, 2625–2658 (1996).
41. Lombardi, A. et al. Retrostructural analysis of metalloproteins: application to
the design of a minimal model for diiron proteins. Proc. Natl Acad. Sci. USA
97, 6298–305 (2000).
42. Summa, C. M., Lombardi, A., Lewis, M. & DeGrado, W. F. Tertiary templates
for the design of diiron proteins. Curr. Opin. Struct. Biol. 9, 500–508 (1999).
43. Lazar, G. A., Desjarlais, J. R. & Handel, T. M. De novo design of the
hydrophobic core of ubiquitin. Protein Sci. 6, 1167–1178 (1997).
44. Di Costanzo, L. et al. Toward the de novo design of a catalytically active helix
bundle: a substrate-accessible carboxylate-bridged dinuclear metal center.
J. Am. Chem. Soc. 123, 12749–57 (2001).
45. Kaplan, J. & DeGrado, W. F. De novo design of catalytic proteins. Proc. Natl
Acad. Sci. USA 101, 11566–11570 (2004).
46. Maglio, O., Nastri, F., Pavone, V., Lombardi, A. & DeGrado, W. F.
Preorganization of molecular binding sites in designed diiron proteins.
Proc. Natl Acad. Sci. USA 100, 3772–3777 (2003).
47. Geremia, S. et al. Response of a designed metalloprotein to changes in metal
ion coordination, exogenous ligands, and active site volume determined by
X‑ray crystallography. J. Am. Chem. Soc. 127, 17266–76 (2005).
48. DeGrado, W. F. et al. Sliding helix and change of coordination geometry in a
model di-MnII protein. Angew. Chem. Int. Ed. 42, 417–420 (2003).
49. Summa, C. M., Rosenblatt, M. M., Hong, J. K., Lear, J. D. & DeGrado, W. F.
Computational de novo design, and characterization of an A2B2 diiron protein.
J. Mol. Biol. 321, 923–938 (2002).
review article
50. Marsh, E. N. & DeGrado, W. F. Noncovalent self-assembly of a heterotetrameric
diiron protein. Proc. Natl Acad. Sci. USA 99, 5150–5154 (2002).
51. Papoian, G. A., DeGrado, W. F. & Klein, M. L. Probing the configurational
space of a metalloprotein core: an ab initio molecular dynamics study of Duo
Ferro 1 binuclear Zn cofactor. J. Am. Chem. Soc. 125, 560–569 (2003).
52. Cochran, F. V. et al. Computational de novo design and characterization of a
four-helix bundle protein that selectively binds a nonbiological cofactor. J. Am.
Chem. Soc. 127, 1346–1347 (2005).
53. Nanda, V. et al. De novo design of a redox-active minimal rubredoxin mimic.
J. Am. Chem. Soc. 127, 5804–5805 (2005).
54. Clarke, N. D. & Yuan, S. M. Metal search: a computer program that helps
design tetrahedral metal-binding sites. Proteins 23, 256–263 (1995).
55. Klemba, M., Gardner, K. H., Marino, S., Clarke, N. D. & Regan, L. Novel
metal-binding proteins by design. Nature Struct. Biol. 2, 368–373 (1995).
56. Regan, L. & Clarke, N. D. A tetrahedral zinc(II)-binding site introduced into a
designed protein. Biochemistry 29, 10878–10883 (1990).
57. Hellinga, H. W. & Richards, F. M. Construction of new ligand binding sites in
proteins of known structure. I. Computer-aided modeling of sites with pre-
defined geometry. J. Mol. Biol. 222, 763–785 (1991).
58. Marvin, J. S. & Hellinga, H. W. Conversion of a maltose receptor into
a zinc biosensor by computational design. Proc. Natl Acad. Sci. USA
98, 4955–4960 (2001).
59. Telmer, P. G. & Shilton, B. H. Structural studies of an engineered zinc biosensor
reveal an unanticipated mode of zinc binding. J. Mol. Biol. 354, 829–840 (2005).
60. Benson, D. E., Wisz, M. S. & Hellinga, H. W. Rational design of nascent
metalloenzymes. Proc. Natl Acad. Sci. USA 97, 6292–6297 (2000).
61. Pinto, A. L., Hellinga, H. W. & Caradonna, J. P. Construction of a catalytically
active iron superoxide dismutase by rational protein design. Proc. Natl Acad.
Sci. USA 94, 5562–5567 (1997).
62. Bolon, D. N. & Mayo, S. L. Enzyme-like proteins by computational design.
Proc. Natl Acad. Sci. USA 98, 14274–14279 (2001).
63. Desmet, J., Demaeyer, M., Hazes, B. & Lasters, I. The dead-end
elimination theorem and its use in protein side-chain positioning. Nature
356, 539–542 (1992).
64. Looger, L. L. & Hellinga, H. W. Generalized dead-end elimination algorithms
make large-scale protein side-chain structure prediction tractable: implications
for protein design and structural genomics. J. Mol. Biol. 307, 429–445 (2001).
65. Host, G. E., Razkin, J., Baltzer, L. & Jonsson, B. H. Combined enzyme and
substrate design: grafting of a cooperative two-histidine catalytic motif
into a protein targeted at the scissile bond in a designed ester substrate.
ChemBioChem 8, 1570–1576 (2007).
66. Kuhlman, B. & Baker, D. Native protein sequences are close to optimal for
their structures. Proc. Natl Acad. Sci. USA 97, 10383–10388 (2000).
67. Jiang, L. et al. De novo computational design of retro-aldol enzymes. Science
319, 1387–1391 (2008).
68. Rothlisberger, D. et al. Kemp elimination catalysts by computational enzyme
design. Nature 453, 190–195 (2008).
69. Tanaka, F., Fuller, R., Shim, H., Lerner, R. A. & Barbas, C. F. Evolution of
aldolase antibodies in vitro: correlation of catalytic activity and reaction-based
selection. J. Mol. Biol. 335, 1007–1018 (2004).
70. Tantillo, D. J., Chen, J. & Houk, K. N. Theozymes and compuzymes: theoretical
models for biological catalysis. Curr. Opin. Chem. Bio. 2, 743–750 (1998).
71. Zanghellini, A. et al. New algorithms and an in silico benchmark for
computational enzyme design. Protein Sci. 15, 2785–2794 (2006).
72. Ladman, Y., Schwartz, J. T. & Wolfson, H. J. Affine invariant model-based
object recognition. IEEE Trans. Robot. Automat. 6, 578–589 (1990).
73. Wolfson, H. J. & Rigoutsos, I. Geometric hashing: an overview. IEEE Comp.
Sci. Eng. 4, 10–21 (1997).
74. Li, H., Helling, R., Tang, C. & Wingreen, N. Emergence of preferred structures
in a simple model of protein folding. Science 273, 666–669 (1996).
75. Li, H., Tang, C. & Wingreen, N. S. Are protein folds atypical? Proc. Natl Acad.
Sci. USA 95, 4987–4990 (1998).
76. Hilvert, D. Critical analysis of antibody catalysis. Annu. Rev. Biochem.
69, 751–792 (2000).
77. Thorn, S. N., Daniels, R. G., Auditor, M. T. & Hilvert, D. Large rate
accelerations in antibody catalysis by strategic use of haptenic charge. Nature
373, 228–230 (1995).
78. Hollfelder, F., Kirby, A. J. & Tawfik, D. S. Off‑the‑shelf proteins that rival
tailor-made antibodies as catalysts. Nature 383, 60–62 (1996).
79. Warshel, A. Electrostatic origin of the catalytic power of enzymes and the role
of preorganized active sites. J. Biol. Chem. 273, 27035–27038 (1998).
80. Voigt, C. A., Mayo, S. L., Arnold, F. H. & Wang, Z. G. Computational method to
reduce the search space for directed protein evolution. Proc. Natl Acad. Sci. USA
98, 3778–3783 (2001).
81. Voigt, C. A., Martinez, C., Wang, Z. G., Mayo, S. L. & Arnold, F. H.
Protein building blocks preserved by recombination. Nature Struct. Biol.
9, 553–558 (2002).
24 nature chemistry | VOL 2 | JANUARY 2010 | www.nature.com/naturechemistry
© 2010 Macmillan Publishers Limited. All rights reserved.
Nature chemistry doi: 10.1038/nchem.473
82. Treynor, T. P., Vizcarra, C. L., Nedelcu, D. & Mayo, S. L. Computationally
designed libraries of fluorescent proteins evaluated by preservation and
diversity of function. Proc. Natl Acad. Sci. USA 104, 48–53 (2007).
83. Lippow, S. M., Wittrup, K. D. & Tidor, B. Computational design of antibody-
affinity improvement beyond in vivo maturation. Nature Biotechnol.
25, 1171–1176 (2007).
84. Radzicka, A. & Wolfenden, R. A proficient enzyme. Science 267, 90–93 (1995).
85. Lienhard, G. E. Enzymatic catalysis and transition-state theory. Science
180, 149–154 (1973).
86. Kraut, D. A., Carroll, K. S. & Herschlag, D. Challenges in enzyme mechanism
and energetics. Annu. Rev. Biochem. 72, 517–571 (2003).
87. Jencks, W. P. Catalysis in Chemistry and Enzymology (McGraw-Hill, 1969).
88. Noy, D., Moser, C. C. & Dutton, P. L. Design and engineering of
photosynthetic light-harvesting and electron transfer using length, time, and
energy scales. Biochim. Biophys. Acta 1757, 90–105 (2006).
89. Nagel, Z. D. & Klinman, J. P. Tunneling and dynamics in enzymatic hydride
transfer. Chem. Rev. 106, 3095–3118 (2006).
90. Bruice, T. C. Computational approaches: Reaction trajectories, structures,
and atomic motions. Enzyme reactions and proficiency. Chem. Rev.
106, 3119–3139 (2006).
91. Boehr, D. D., McElheny, D., Dyson, H. J. & Wright, P. E. The dynamic energy
landscape of dihydrofolate reductase catalysis. Science 313, 1638–1642 (2006).
92. Gerstein, M., Lesk, A. M. & Chothia, C. Structural mechanisms for domain
movements in proteins. Biochemistry 33, 6739–6749 (1994).
93. Henzler-Wildman, K. A. et al. A hierarchy of timescales in protein dynamics is
linked to enzyme catalysis. Nature 450, 913–916 (2007).
94. Henzler-Wildman, K. A. et al. Intrinsic motions along an enzymatic reaction
trajectory. Nature 450, 838–844 (2007).
95. Xiang, J. Y., Jung, J. Y. & Sampson, N. S. Entropy effects on protein hinges:
The reaction catalyzed by triosephosphate isomerase. Biochemistry
43, 11436–11445 (2004).
96. Munro, A. W. et al. P450BM3: the very model of a modern flavocytochrome.
Trends Biochem. Sci. 27, 250–257 (2002).
97. Wyman, J. & Gill, S. J. Binding and Linkage (University Science Books, 1990).
98. Anderson, J. L. R., Koder, R. L., Moser, C. C. & Dutton, P. L. Controlling
complexity and water penetration in functional de novo protein design.
Biochem. Soc. Trans. 36, 1106–1111 (2008).
99. Joyce, G. F. The antiquity of RNA-based evolution. Nature
418, 214–221 (2002).
100. Doudna, J. A. & Lorsch, J. R. Ribozyme catalysis: not different, just worse.
Nature Struct. Mol. Biol. 12, 395–402 (2005).
101. Lee, S. & Jung, S. Cyclosophoraose as a catalytic carbohydrate for
methanolysis. Carbohydr. Res. 339, 461–468 (2004).
102. Hill, D. J., Mio, M. J., Prince, R. B., Hughes, T. S. & Moore, J. S. A field guide to
foldamers. Chem. Rev. 101, 3893–4011 (2001).
103. Goodman, C. M., Choi, S., Shandler, S. & DeGrado, W. F. Foldamers as
versatile frameworks for the design and evolution of function. Nature Chem.
Biol. 3, 252–262 (2007).
104. Nanda, V. & DeGrado, W. F. Computational design of heterochiral peptides
against a helical target. J. Am. Chem. Soc. 128, 809–816 (2006).
105. Nanda, V. & Degrado, W. F. Simulated evolution of emergent chiral structures
in polyalanine. J. Am. Chem. Soc. 126, 14459–14467 (2004).
106. Baldauf, C., Gunther, R. & Hoffmann, H.‑J. Helix Formation in α, - and β,
γ-hybrid peptides: Theoretical insights into mimicry of α- and β-peptides.
J. Org. Chem. 71, 1200–1208 (2006).
107. Sandvoss, L. M. & Carlson, H. A. Conformational behavior of β-proline
oligomers. J. Am. Chem. Soc. 125, 15855–15862 (2003).
108. Lee, O.‑S. & Saven, J. G. Simulation studies of a helical m‑phenylene ethylene
foldamer. J. Phys. Chem. B 108, 11988–11994 (2004).
109. Smaldone, R. A. & Moore, J. S. Reactive sieving with foldamers:
inspiration from nature and directions for the future. Chem. Eur. J.
14, 2650–2657 (2008).
110. Blake, C. C. et al. Structure of hen egg-white lysozyme. A three-dimensional
Fourier synthesis at 2 Å resolution. Nature 206, 757–761 (1965).
111. Nanda, V. Do‑it‑yourself enzymes. Nature Chem. Biol. 4, 273–275 (2008).
Acknowledgements
VN acknowledges support from the NIH Director’s New Innovator Award Program,
1-DP2-OD006478-01 and the NSF BMAT program DMR-0907273. RLK acknowledges
supported by the following grants: MCB-0920448 from the NSF, MCB-5G12 RR03060
toward support for the NMR facilities at the City College of New York, P41 GM-66354
to the New York Structural Biology Center and infrastructure support from NIH 5G12
RR03060 from the National Center for Research Resources.
Author information
The authors declare no competing financial interests.